Glycoside Hydrolase Family 77

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Contents

Substrate specificities

Glycoside hydrolase family GH77 is a member of the α-amylase clanGH-H [1], together with GH13 and GH70 [2]. The family is monospecific with the 4-α-glucanotransferase (EC 2.4.1.25), that is known as disproportionating enzyme (D-enzyme) in plants [3] or amylomaltase in bacteria [4] and archaeons [5]. Around 2,000 members [6] are known that originate mostly from Bacteria; the family also contains a few tens of additional sequences from each Archaea and Eucarya (plants and green algae). Only slightly above 1% of the family members have been biochemically characterized [6].

Amylomaltase catalyses glucan-chain transfer from one α-1,4-glucan to another α-1,4-glucan (or to 4-hydroxyl group of glucose) or within a single linear glucan molecule to produce a cyclic α-1,4-glucan with degree of polymerization starting from 17 [3, 4, 5]. Cyclodextrin glucanotransferase, a member of the α-amylase family GH13, also produces cyclic α-1,4-glucans, but with a small degree of polymerization (6-8), called cyclodextrins [7].

Kinetics and Mechanism

Reaction products have been analysed for several family GH77 enzymes by TLC (mainly) and HPLC, including the D-enzyme from potato [3] as well as amylomaltases from Clostridium butyricum [9], Thermus aquaticus [4], Aquifex aeolicus [10], Pyrobaculum aerophilum [5], Thermus thermophilus [11] and Borrelia burgdorferi [12], but the kinetics were determined only for a few, e.g., [5, 10, 11].

Catalytic Residues

The family GH77 4-α-glucanotransferases fold into a (β/α)8-barrel with the catalytic machinery consisting of a strand β4-aspartic acid (catalytic nucleophile), β5-glutamic acid (proton donor) and β7-aspartic acid (transition-state stabilizer). For example these are Asp293, Glu340 and Asp395 in the amylomaltase from Thermus aquaticus [13]. The somewhat unusual conformations exhibited mainly by the supposed catalytic nucleophile (Asp293) have been explained by the high experimental pH of 9.0 used during crystallization [14]. The catalytic triad is supported by a later site-directed mutagenesis study [11]. All the family GH77 4-α-glucanotransferases share the 4-7 conserved sequence regions [12, 15] characteristic for the α-amylase clanGH-H [16].

Three-dimensional structures

Five 3-D structures have been solved for the following family GH77 members: (i) the amylomaltases from Thermus aquaticus [13], Aquifex aeolicus (unpublished; PDB ID 1tz7), Thermus thermophilus [17] and Thermus brockianus [18]; and (ii) the D-enzyme from potato (unpublished; PDB ID 1x1n). The crystallization of the amylomaltase from Corynebacterium glutamicum has also been reported [19].

The main structural feature that discriminates the family GH77 amylomaltases from typical α-amylase family GH13 members is the lack of domain C [13] that succeeds the catalytic (β/α)8-barrel (TIM-barrel) in the family GH13 enzymes. The eight-fold symmetry of the catalytic barrel in the family GH77 is disrupted by several insertions between the barrel β-strands that form the so-called subdomains B1, B2 and B3 [13]. Subdomain B1 consists of a highly twisted four-stranded antiparallel β-sheet with two α-helices and is also present in other enzymes from the α-amylase clanGH-H (known as domain B). Subdomain B2 has predominantly an α-helical structure and it is unique to amylomaltases. Subdomain B3 could have a role of domain C from the α-amylase family [13].

Interestingly, primary structures of amylomaltases from borreliae contain unique sequence features [15], i.e. natural mutations in functionally important positions from conserved sequence regions. The most important and remarkable one is represented by otherwise extremely well-conserved and functional arginine in position i-2 with respect to the catalytic nucleophile that is replaced by a lysine [15]. It is worth mentioning that this arginine positioned two residues before the catalytic nucleophile in the conserved sequence region II was considered to belong to the four residues conserved invariantly (along with the catalytic triad) throughout the α-amylase family [16]. Its substitution is therefore of a special interest because the GH77 protein from Borrelia burgdorferi exhibits amylomaltase activity [12]. Since, however, the lysine could play the role of the original arginine, it is not possible to say unambiguously that the catalytic triad alone (aspartic acid, glutamic acid and aspartic acid at strands β4, β5 and β7, respectively, of the catalytic TIM-barrel) is enough for a GH-H protein to be a functional member of the α-amylase family [12]. There are several additional putative amylomaltases from various borreliae available; some of them possess the Arg-to-Lys mutation, indicating the borreliae enzymes may occupy an outstanding position in evolution of this 4-α-glucanotransferase family GH77 [20].

In plants particularly a longer version of D-enzyme (DPE1) was identified and named DPE2 [21, 22]. It was recently described also in certain bacteria [20]. It usually contains a ~140 amino acid residues long insert within the catalytic GH77 TIM barrel and two copies of a starch-binding domain of family CBM20 succeeded by a short coiled coil motif positioned N-terminally [23]. Interestingly, removing the insert leads to inactivation of the DPE2 although the insert itself has nothing to do with the catalytic action of the enzyme [24].

Family Firsts

First stereochemistry determination

There is no direct evidence for the stereochemistry of hydrolysis of the glucosidic bond in the reaction of any family GH77 4-α-glucanotransferase. HPLC analyses of reaction products reported for amylomaltases from, e.g., Aquifex aeolicus [10], Pyrobaculum aerophilum [5] and Thermus thermophilus [11] was used to propose a retaining mechanism assumed in analogy with the mechanism confirmed in α-amylase family GH13.

Asp293, was identified as a catalytic nucleophile in the amylomaltase from Thermus thermophilus on the basis of mutant enzymes (D293N and D293A) that exhibited greatly reduced disproportionation activity [11].

The catalytic proton donor, Glu340, was identified as a catalytic residue in the amylomaltase from Thermus thermophilus by demonstration of a greatly reduced disproportionation activity of the E340Q and E340A mutant enzymes [11].

First 3-D structure

The first 3-D structure of a GH77 member was that of the amylomaltase from Thermus aquaticus solved first as a free enzyme (PDB ID 1cwy) [13] and subsequently also as a complex with acarbose (PDB ID 1esw) [14].